A Freedom of Coxiella Burnetii Infection Survey in European Bison (Bison Bonasus) in Poland

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Communication A Freedom of Coxiella burnetii Infection Survey in European Bison (Bison bonasus) in Poland

Michał K. Krzysiak 1,2,* , Martyna Puchalska 3, Wanda Olech 4 and Krzysztof Anusz 3

1 Białowieza˙ National Park, Park Pałacowy 11, 17-230 Białowieza,˙ Poland 2 Institute of Forest Sciences, Faculty of Civil Engineering and Environmental Sciences, Białystok University of Technology, Wiejska 45 E, 15-351 Białystok, Poland 3 Department of Food Hygiene and Public Health Protection, Institute of Veterinary Medicine, Warsaw University of Life Sciences—SGGW, Nowoursynowska 159, 02-776 Warsaw, Poland; [email protected] (M.P.); [email protected] (K.A.) 4 Department of Animal Genetics and Conservation, Institute of Animal Science, Warsaw University of Life Sciences—SGGW, Ciszewskiego 8, 02-786 Warsaw, Poland; [email protected] * Correspondence: [email protected]

Simple Summary: Q fever is one of the important diseases transmissible from animals to humans. The source of infection can be numerous species of animals including mammals, birds, reptiles, amphibians as well as ticks. The role of wildlife in its epidemiology is poorly understood. Therefore, we examined 523 sera samples obtained from European bison for the presence of speciﬁc antibodies to assess whether infection occurs in this species and whether European bison may be an important source of infection in the natural environment as suggested by historical reports. The antibodies were found only in one free-living bull, while two other samples were doubtful. The results suggest the transmission of infection to the European bison was rather accidental and its role as an important Citation: Krzysiak, M.K.; Puchalska, source of infection nowadays is unlikely. M.; Olech, W.; Anusz, K. A Freedom of Coxiella burnetii Infection Survey Abstract: Q fever is an important zoonosis caused by the intracellular Gram-negative bacteria in European Bison (Bison bonasus) Coxiella burnetii. The source of infection are numerous species of mammals, birds, reptiles and in Poland. Animals 2021, 11, 651. amphibians, as well as ticks. The disease is widespread throughout Europe, but the role of wildlife https://doi.org/10.3390/ani11030651 in its epidemiology is poorly understood. The European bison (Bison bonasus) population has been growing European-wide quite dynamically over the last few years. The aim of this study Academic Editor: was to determine whether C. burnetii infection occurs in European bison and whether it can be David González-Barrio considered an important bacterial reservoir in the natural environment. Five hundred and twenty

Received: 1 February 2021 three samples of European bison sera originating from 14 (out of the 26 existing) Polish populations Accepted: 24 February 2021 were examined for the presence of speciﬁc antibodies using an ID Screen Q Fever Indirect Multi- Published: 1 March 2021 species ELISA test. Only one (0.19%) serum sample was positive in ELISA, and two other samples were doubtful. The only seropositive animal found in this study was a free-living bull. It suggests Publisher’s Note: MDPI stays neutral possible transmission from domestic cattle by sharing pastures. The transmission of C. burnetii into with regard to jurisdictional claims in the European bison was rather accidental in the country and its role as an important wild reservoir published maps and institutional afﬁl- is unlikely. Since no tests are available for wildlife ruminants there is a need for the adaptation of iations. the available tests.

Keywords: Coxiella burnetii; Q fever; serology; epidemiology; wildlife; European bison

Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article 1. Introduction distributed under the terms and Q fever is a widely distributed reproductive disease of ruminants caused by intra- conditions of the Creative Commons cellular bacteria Coxiella burnetii. The pathogen is commonly transmitted to humans Attribution (CC BY) license (https:// and remains an emerging concern for public health. The source of infection are numer- creativecommons.org/licenses/by/ ous species of mammals, birds, reptiles and amphibians. Ticks are important vectors of 4.0/).

Animals 2021, 11, 651. https://doi.org/10.3390/ani11030651 https://www.mdpi.com/journal/animals Animals 2021, 11, 651 2 of 9

C. burnetii transmission; therefore, the climate changes observed in the recent decades may provoke the shift of those arthropods into new locations, increasing the risk of tick-borne infections to occur [1]. In humans, infection as a result of a tick bite is rare, while potential sources of infection may be dusty feces of infected ticks, or exposure to dust with dried secretions of the reproductive system spread by the wind up to a distance of 2 km. Free- living animals may be a reservoir and source of C. burnetii infection for both humans and domestic animals. Q fever is widely spread all over Europe, however the role of wildlife in its transmission is still poorly understood. Dairy cattle are considered the main reservoir in Poland with the animal-level and herd-level seroprevalence around 25% [2]. C. burnetii DNA was detected in 3% of wildlife tested in a survey in the north-west of the country, which included roe deer, red deer and wild boar [3]. In the same study, the presence of bacteria was also confirmed in the ticks Ixodes ricinus from the same area. Other reports also confirmed the infection in wild ruminants, rabbits, rodents, foxes and ticks associated with their sylvatic ecosystems [4,5]. Moreover, specific antibodies were found in wild ranging cervids and mouflon [6,7]. European bison are the largest herbivores in Europe. Their conservation strategies should also include the monitoring of zoonotic diseases as an element of the One Health concept. However, Q fever studies have been neglected for the last two decades. The first report on the occurrence of C. burnetii in European bison dates back to the nineteen-eighties, when the exposure of forty-seven free-ranging European bison was investigated from Borecka forest (54◦5018.09” N 21◦55021.467” E) in connection to a Q fever epidemic in local cattle herds. The presence of specific antibodies was then confirmed in 76% of animals by the microagglutination test and complement fixation test (CFT) [8]. Furthermore, earlier in 1980–1983, a natural foci of Q fever could have been located in the Białowieska forest (52◦4209.861” N 23◦5104.52” E) [9], where the conservation of the endangered species after their complete extinction from the wild was initiated. Additionally, transmission to humans was suspected as 10% of European bison caretakers from Białowieza˙ National Park (52◦4209.861” N 23◦5104.52” E) became infected [8]. Further studies excluded exposure of European bison to C. burnetii by serological testing in 122 European bison from Białowieska forest in years 1991–2001 [10,11]. The population of the species, which remains listed by the International Union for Conservation (IUCN) of Nature’s Red List of Threatened Species increases successfully thanks to many national and international projects, reaching already \ total of over 8400 individuals. They are most often reared in the enclosures but wild European bison population with frequent contact with people and domestic animals significantly increases [12]. In 2019, 74% of European bison were free-living [12]. Taking into account abovementioned, the aim of this study was to determine whether C. burnetii infection occurs in the European bison and whether it can be considered an important bacterial reservoir in the natural environment.

2. Materials and Methods 2.1. Study Design To calculate the representative number of European bison in each of the populations to substantiate a prevalence of 1.5% or below with the overall α error set to 0.05, the FreeCalc software of Epitools [13,14] was used. The mean population size was determined by Euro- pean bison Pedigree Books (2013–2018). The targeted population size number increased from 1196 animals in 2013 to 1478 in 2018 with the mean value of 1377 individuals [12]. Considering the design prevalence at 1%, test sensitivity and speciﬁcity of about 100% and desired type I and II errors at 0.05, the targeted sample size for the whole population tested was 267. However, for the smaller herds, all the animals or all the available samples were included in the study. The samples were collected during the monitoring of European bison health in the years 2011–2017 as a part of the scientiﬁc cooperation of National Veterinary Research Institute in Puławy (NVRI) with herd/population managers based on: the opinion of the Minister of the Environment of 27 October 2014 (ZOP/06-061/51/2014); the Decision of the General Di- Animals 2021, 11, 651 3 of 9 Animals 2021, 11, 651 3 of 9

General Director for Environmental Protection of December 31, 2014 (DZP- rectorWG.6401.06.23.2014.km.2); for Environmental Protection individual of 31 permits December of the 2014 Director (DZP-WG.6401.06.23.2014.km.2); of the Białowieża National individualPark of December permits of20, the 2013 Director (PN/061/22/2013) of the Białowie and za˙May National 15, 2017 Park (PN of/061/14/2017) 20 December and 2013 per- (PN/061/22/2013)mits of the Kobiór and Forest 15 MayDistrict 2017 (ZG (PN/061/14/2017)-7326 (1) -9/2014). and Since permits 2017, ofthe the sample Kobió collectionr Forest Districthas been (ZG-7326 carried (1) out -9/2014). as part of Since the 2017,project the “Complex sample collection project of has European been carried bison out conserva- as part oftion the by project State “Complex Forests” financed project of by European the Forest bison Fund conservation (Poland) in by accordance State Forests” wit ﬁnancedh contract byNo. the OR.271.3.10.2017. Forest Fund (Poland) in accordance with contract No. OR.271.3.10.2017. AA total total of of 523 523 serum serum samples samples were were collected collected from from European European bison bison from from 14 14 (out (out of of 26 26 existing)existing) different different populations populations spread spread across across the countrythe country including including wild-ranging wild-ranging European Euro- bisonpean from bison Białowieska from Białowieska (including (including Białowie Białowieżaza˙ National Natio Park)nal Park) (n = 170), (n = Borecka170), Borecka (n = 37)(n = and37) Knyszy´nska(and Knyszyńskan = 52) (n = forests 52) forests and several and several other other herds herds kept inkept captivity in captivity between between 2013 and 2013 2018.and The2018. distribution The distribution and numbers and numbers of the studied of the Europeanstudied European bison are bison presented are presented in Figure1 .in TheFigure samples 1. The originated samples bothoriginated from female both from (n = 286)female and (n male = 286) (n =and 219) male European (n = 219) bison European aged betweenbison aged 5 days between and 29 years.5 days Samples and 29 wereyears. taken Samples from pharmacologicallywere taken from pharmacologically immobilized (for placingimmobilized collars (for with placing telemetric collars transmitters with telemetric or diagnostic transmitters reasons), or diagnostic fallen or euthanized reasons), fallen due toor poor euthanized health individuals due to poor in health accordance individuals with the in correspondingaccordance with decisions the corresponding of the Minister deci- ofsions the Environment of the Minister and ofthe theGeneral Environment Director and for the Environmental General Director Protection. for Environmental The blood was Pro- collectedtection. immediatelyThe blood was after collected immobilization immediately or culling after immobilization through the puncture or culling of the through external the jugular vein (vena iugularis externa), less often from the tail vein (vena caudalis mediana). puncture of the external jugular vein (vena iugularis externa), less often from the tail vein Blood from dead, necropsied animals was collected in the form of a clot from the heart or (vena caudalis mediana). Blood from dead, necropsied animals was collected in the form of from body cavities. The decayed or extensively hemolyzed samples were excluded from a clot from the heart or from body cavities. The decayed or extensively hemolyzed sam- the study to minimize the risk of false result. Blood was collected into the sterile 7–9 mL ples were excluded from the study to minimize the risk of false result. Blood was collected tubes, centrifuged within 24 h, and the obtained serum was frozen at −70 ◦C until analysis into the sterile 7–9 mL tubes, centrifuged within 24 h, and the obtained serum was frozen in the sample bank of the Department of Virology, NVRI, Poland. at −70 °C until analysis in the sample bank of the Department of Virology, NVRI, Poland.

FigureFigure 1. 1.The The map map of of Poland Poland showing showing the the numbers numbers of of European European bison bison divided divided into into free-ranging free-ranging pop- ulationspopulations (green) (green) and captive and captive herds herds (black). (black). The red The cross red indicatescross indicates the location the location of single of seropositive,single sero- 6-year-oldpositive, 6 male-year from-old ma wildle populationfrom wild population of Białowieska of Białowieska Forest. Forest.

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2.2. ELISA (Enzyme-Linked Immunosorbent Assay) Serum samples were tested for presence of antibodies to Coxiella burnetii using commer- cial ELISA test ID Screen Q Fever Indirect Multi-species (IDvet, Grabels, France). This kit is based on a mix of phase I and II antigens obtained after the purification and inactivation of a C. burnetii strain isolated from the placenta of an aborting cow. The test was performed according to the manufacturer’s instructions. Briefly, tested and control sera (negative control—NC and positive control—PC) were diluted 1:50 in Dilution Buffer 2 at the dilution plate and then, 100 µL of the diluted sera were transferred to the test plate. The plate was incubated for 45 ± 4 min at 21 ◦C(±5 ◦C) and washed 3 times with the washing solution. A conjugate working solution was prepared by diluting the concentrated Conjugate (10×) in Dilution Buffer 3 in a ratio of 1:10. An amount of 100 µL of conjugate working solution was added to all wells. The plates were incubated for 30 ± 3 min at 21 ◦C(±5 ◦C) and then washed 3 times with the washing fluid solution. An amount of 100 µL of Substrate solution was added to all wells and the plates were incubated in a dark place for 15 ± 2 min at 21 ◦C (±5 ◦C). The reaction was stopped by adding 100 µL Stop Solution to each well. The optical densities (OD) were read at 450 nm using an ELISA plate reader (Epoch, BioTek, Winooski, VT). The results were interpreted by calculation of the Sample to Positive percentage (S/P%) = (ODsample−ODNC) × as S/P% (ODPC−ODNC) 100 The result was considered reliable if: the average ODPC value is greater than 0.350 and the ratio of the average ODPC value and the average ODNC value is greater than 3. If S/P% ≤ 40%, the result was considered negative; if 40% < S/P% ≤ 50%, the result was considered doubtful; if S/P% > 50%, the result was considered positive.

2.3. Statistical Analysis For statistical evaluation, STATA software version 11 (StataCorp., College Station, TX, USA) was used. The percentage of C. burnetii seropositive animals and 95% conﬁdence interval (CI) values were calculated using binomial exact test. The S/P values were analyzed by kernel estimated frequency plot. The univariate associations between the seropositivities to C. burnetii, environmental (origin, population type, health status) and individual-level (sex, age) variables were estimated using Fisher’s exact test.

3. Results Only one (0.19%; 95% CI: 0.005–1.1) serum sample reacted positively in the ELISA. It derived form 6-year-old European bison bull (No 1983, born in 2007) culled due to poor body condition and severe balanoposititis from the free-living population in January 2013. Furthermore, two other samples were doubtful. These included also free-living males: a 3- month-old calf (No 2250; born in 2012) and 6-year-old bull (No 1984; born in 2007), which were also eliminated by culling from Białowieska forest in 2013. The results in relation to different variables are presented at Table1. Animals 2021, 11, 651 5 of 9

Table 1. Descriptive statistics of seropositive to Coxiella burnetii European bison (Bison bonasus) with regard to their different characteristics.

Number Positive/Examined % (95% Confidence Interval) Location (Global Positioning System) (N * = 523) Bałtów (51◦103.759” N 21◦ 32030.098” E) 0/7 0 (0–41.0) Białowieska forest (52◦4209.861” N 23◦5104.52” E) 1/171 ** 0.58 (0.1–3.2) Bieszczady mountains (49◦7012.898” N 22◦4500.782” E) 0/24 0 (0–14.2) Borecka forest (54◦5018.09” N 21◦55021.467” E) 0/37 0 (0–9.5) Gołuchów (51◦50058.047” N 17◦55050.863” E) 0/12 0 (0–26.5) Kiermusy (53◦1207.699” N 22◦42044.245” E) 0/2 0 (0–84.2) Knyszyńskaforest (53◦15035.036” N 23◦38037.11” E) 0/52 0 (0–6.8) Niepołomice (50◦1045.67” N 20◦20046.365” E) 0/49 0 (0–7.2) Pszczyna (49◦58029.284” N 18◦55052.465” E) 0/92 0 (0–3.9) Smardzewice (51◦28039.975” N 20◦300.964” E) 0/60 0 (0–6.0) Strzelinko (54◦31045.236” N 16◦56058.023” E) 0/1 0 (0–97.5) Ustroń(49◦42058.955” N 18◦49059.765” E) 0/2 0 (0–84.2) ZOO Łód´z(51◦45039.629” N 19◦24045.333” E) 0/3 0 (0–70.8) ZOO Warsaw (52◦15028.9” N 21◦1021.035” E) 0/11 0 (0–28.5) Population type (N = 523) free-living 1/179 ** 0.56 (0.1–3.1) captive 0/344 0 (0–1.1) Gender (N = 505) female 0/281 0 (0–1.3) male 1/224 ** 0.44 (0.01–2.4) Age group (N = 474) ≤1 year old 0/98 *** 0 (0–3.7) 2–3 years old 0/114 0 (0 = 3.2) ≥4 years old 1/262 *** 0.38 (0.1–2.1) Health status (N = 501) immobilized (apparently healthy) 0/348 0 (0–1.1) eliminated (by culling) 1/134 ** 0.74 (0.02–4.1) fallen 0/15 0 (0–21.8) traffic accident 0/4 0 (0–60.2) * number of examined in the category (taking into account the missing data); ** two and *** one additional doubtful result was detected.

The distribution of S/P% with a single peak skewed towards negative values may be observed in Figure2. AnimalsAnimals 20212021, 11, 11, 651, 651 6 6of of 9 9

FigureFigure 2. 2. DotblotDotblot distribution distribution of of Sample Sample to to Positive Positive percentage percentage (S/P% (S/P%)) values values of ofID ID Screen Screen Q QFever Fever IndirectIndirect Multi Multi-species-species (IDvet, (IDvet, Grabels, Grabels, France) France) testing testing for for the the presence presence of of CoxiellaCoxiella burnetii burnetii antibod-antibodies iesin in 523 523 sera sera of of European European bison bison (Bison (Bison bonasus bonasus) collected) collected between between 2013 2013 and and 2018 2018 in 14 in different 14 different locations locationsin Poland. in Poland.

4.4. Discussion Discussion Nowadays,Nowadays, the the increasing increasing European European bison bison population population size size and and density, density, decreasing decreasing wildwild habitat habitat and and introduction introduction strategies strategies induces induces more more frequent frequent contact contact with with other other wild wild reservoirsreservoirs and and domestic domestic animals atat thethe shrinkingshrinking interface. interface. The The population population size size of Europeanof Euro- peanbison bison in Poland in Poland and inand Europe in Europe has tripled has tripled in the in last the twenty last twenty years [years12]. Moreover, [12]. Moreover, already alreadythree quarters three quarters of the animalsof the animals are free are ranging free ranging including including in several in several new locations new locations where wherethe species the species was introduced was introduced or re-introduced or re-introduced after the after extinction the extinction in Europe in Europe [12]. The [12]. role The of rolethe of species the species as a reservoir as a reservoir of the zoonotic of the zoonotic bacteria bacteria at present at waspresent therefore was therefore investigated investi- using gatedrepresentative using representative numbers of animalsnumbers in of the animals largest Polishin the populationslargest Polish as populations well as in the as smaller, well ascaptive in the smaller, herds. captive herds. InIn our our study, study, thethe onlyonly seropositiveseropositive animal animal was was a free-livinga free-living bull. bull. In 1980–1983In 1980–1983 the stud-the studiesies of Europeanof European bison bison culled culled at thatat that time time revealed revealed antibodies antibodies to C.to C. burnetii burnetiiin in a 2-year-olda 2-year- oldheifer. heifer. It It was was thought thought toto bebe associated with with the the presence presence of of a natural a natural foci foci of Q of fever Q fever in thein Białowieska the Białowieska forest forest [9]. [The9]. Theoutbreak outbreak was was probably probably associated associated with with the theoccurrence occurrence of Qof fever Q fever epizo epizooticotic outbreaks outbreaks in cattle in cattle and andsheep sheep in neighboring in neighboring villages villages [9]. However, [9]. However, de- spitedespite continuous continuous occurrence occurrence of Q offever Q feverin the indomestic the domestic ruminants ruminants in 1980 in–1983 1980–1983,, no trans- no missiontransmission to European to European bison was bison observed. was observed. European European bison males bison usually males wander usually solitarily wander orsolitarily in small ormale in smallgroups male often groups approaching often approaching farmland and farmland villages, andwhere villages, they can where become they incanto contact become with into domestic contact with animals domestic and humans. animals Meanwhile and humans., females Meanwhile, with calves females and with ju- calves and juveniles create so-called mixed groups, which rarely leave the forest, except veniles create so-called mixed groups, which rarely leave the forest, except for in winter, for in winter, when they may come out onto fields or winter-feeding places in search for when they may come out onto fields or winter-feeding places in search for food. There- food. Therefore, it can be assumed that the exposure of C. burnetii in free-living bull in our fore, it can be assumed that the exposure of C. burnetii in free-living bull in our study may study may have been associated with transmission of pathogen from domestic ruminants have been associated with transmission of pathogen from domestic ruminants by sharing by sharing pastures. In the eighties, due to the occurrence of Q fever in domestic animals pastures. In the eighties, due to the occurrence of Q fever in domestic animals in north- in north-eastern Poland, forty-seven free-ranging European bison from Borecka forest eastern Poland, forty-seven free-ranging European bison from Borecka forest were exam- were examined for the presence of specific antibodies by the microagglutination test and ined for the presence of specific antibodies by the microagglutination test and comple- complement fixation test (CFT). The high seroprevalence (76%) of Coxiella burnetii found ment fixation test (CFT). The high seroprevalence (76%) of Coxiella burnetii found in Euro- in European bison suggested that they could be considered as a potential reservoir of Q pean bison suggested that they could be considered as a potential reservoir of Q fever [8]. Animals 2021, 11, 651 7 of 9

fever [8]. Szarek et al. [15] have linked C. burnetii infection to the pathomorphological changes of heart and kidneys specific for Q fever observed in those studied European bison. Moreover, European bison–human transmission was suspected, since the infection was also confirmed in 10% of the employees of the Białowieza˙ National Park (BNP) staff. However, further studies suggested that the Q fever foci were self-limited in the following years [10,11]. The results of our studies follow this trend. The results of our studies, however, suggest that the exposure of European bison to C. burnetii is rather accidental if even occurring, which may be surprising, as the infections are quite common in cattle in the country [2]. Assuming that the positive result in only one free-living bull was not a false positive reaction, a transmission from cattle would be suspected. The seroprevalence of C. burnetii in cattle in the Podlaskie province, where two major European bison free-living populations of Białowieska and Knyszyńskaforests are living reaches 48% [2]. The density of cattle in the region is also the highest in the country with the intensive milk production and increasing pasture grazing [16]. These findings suggest low susceptibility of European bison to C. burnetii infection despite the potential increasing risk with increasing contacts with domestic Q fever reservoirs in endemic areas. The few reports on Q fever occurrence in wild ruminants in Europe suggest low seroprevalence of C. burnetii, and therefore their role in the transmission and maintain- ing the bacteria in the natural environment is rather doubtful. Low seroprevalences of C. burnetii were reported previously in wild ranging cervids and mouflon [6,7]. Higher seroprevalences were found in farmed deer, which may suggest increased exposure to C. burnetii connected to higher density of animals [17]. However, the factors modulating the risk of exposure to C. burnetii in wildlife are quite complex and should be evaluated individually [18]. Our study together with previous concerning C. burnetii infections in European bison [9,11] also suggest that they may be accidental hosts for the pathogen. Nevertheless, since C. burnetii may cause large reproductive loses to domestic ungulates, we should be cautious when it comes to endangered species as European bison and monitor the epidemic situation. According to OIE [World Organisation for Animal Health; 19], at present, no gold standard technique is available in diagnosis of Q fever. Serological ELISA and direct detection and quantification by PCR should be considered as the methods of choice. Serological analyses may be carried out using ELISA, a complement fixation test (CFT) or indirect immunofluorescence assay (IFA). Among serological methods, ELISA is recommended for routine serological testing of animals for Q fever because it has a high sensitivity and a good specificity, and it is convenient for large-scale screening. The relative sensitivity is the lowest for CFT [19]. An antibody ELISA was used in our research. Some authors question the high test performance in other than small ruminants [20]; however, the assay was successfully used in Q fever epidemiological studies in other species [21]. In general, ELISA also shows improved sensitivity and specificity in relation to the complement fixation test [22,23], despite its performance possibly varying between different manufacturers, different antigens used for plate coating and the species to which it is applied [24]. The report on Q fever cases in European bison in the 1980s was based on CFT [8]; therefore, it could explain why further studies, including present, have differed on C. burnetii exposure in the species, despite potentially increasing risk. Studies on detecting antibodies against C. burnetii demonstrate only previous exposure to the pathogen, not current shedding of bacteria but they can be useful for epidemiological analysis of endemic areas [2,25]. Moreover, seropositivity without detection of pathogen in the sera may be indicative of chronic exposures to C. burnetii [26]. Moreover, some shedders of C. burnetii may be seronegative [27]. Therefore, some discrepancies in the results of serological versus PCR tests carried out on the same animals may be observed. Knap et al. [25] have detected the presence of specific antibodies in the sera of 60 out of 150 (40.4%) animals (cattle, sheep), while the presence of pathogen DNA was confirmed only in 14 out of 150 (9.3%) blood samples. Furthermore, Bellabidi et al. [21] found antibodies to C. burnetii in 75.54% of tested samples of camel sera and no DNA of the pathogen in sera of 138 seropositive animals. It is Animals 2021, 11, 651 8 of 9

noteworthy that there are differences in the shedding of bacteria in different animal species. Cattle shed the bacteria almost exclusively in milk, goats mostly in milk with a minority shedding it in vaginal mucous or feces and sheep in feces, vaginal mucous and milk [27]. Since no tests dedicated to speciﬁc species, except for cattle, are recommended, and no tests are available for wildlife ruminants, the adaptation and optimization of the available test should be considered in further studies [5,18,23,24,28]. Since Q fever may be a potential emerging disease in the areas where contacts between wildlife and humans or domestic animals increase, the need for a reliable test for monitoring of wildlife is growing. Frosinski et al. [29] have also discussed the need for cut-off adaptation, which may be beneﬁcial to obtaining good quality prevalence data. In our study, some samples gave slightly higher S/P% value approaching the cut-off value for doubtful results. However, optimizing cut-off value would require testing a higher number of known C. burnetii seropositive samples collected from the species which are not available at the moment (Figure2). Further studies are needed.

5. Conclusions The only seropositive European bison found in this study was a free-living bull which suggests possible transmission from domestic cattle by sharing pastures; however, a false positive result may be also suspected. The transmission of C. burnetii into the European bison was rather accidental in the country and its role as an important wild reservoir is questionable. Since no tests are available for wildlife ruminants there is a need for the adaptation of the available tests in order to monitor possible C. burnetii circulation in the sylvatic environment.

Author Contributions: Conceptualization, M.K.K. and M.P.; formal analysis, M.K.K.; investigation, M.P. and M.K.K.; resources, M.K.K.; writing—original draft preparation, M.K.K. and M.P.; writing— review and editing, K.A., M.K.K. and M.P.; funding acquisition, W.O. and M.K.K. All authors have read and agreed to the published version of the manuscript. Funding: This work is part of the project “Complex project of European bison conservation by State Forests”, which is financed by the Forest Found (Poland), contract number OR.271.3.10.2017. Institutional Review Board Statement: Not applicable. Data Availability Statement: Raw data are available upon request from the corresponding author. Acknowledgments: The authors want to thank Magdalena Larska, National Veterinary Research Institute, Puławy, Poland for providing samples for testing and statistical analysis. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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